Further evaluation of a modified micronucleus assay with V79 cells for detection of aneugenic effects.

In an earlier publication, we reported on the development of a modified micronucleus assay with V79 cells enabling preferential detection of aneugen-induced micronuclei (Seelbach et al., 1993). Here we present a further evaluation of the modified micronucleus assay based on the investigation of seven further suspected aneugens. Five compounds gave positive results: cadmium chloride, chloral hydrate, hydroquinone, thimerosal and vinblastine. Econazole and pyrimethamine were negative. Up to now, our experience has shown that data produced by the modified V79/micronucleus assay are quite reliable: the variation of spontaneous micronucleus frequencies was low (0.8-1.7%) and the reproducibility of the data was good.

Development of a modified micronucleus assay in vitro for detection of aneugenic effects.

A simple and rapid micronucleus (MN) assay in vitro has been developed with V79 cells enabling preferential detection of aneugen-induced MN. V79 cells were treated with test compounds for 3 hr, the mitoses were shaken off and transferred into new flasks, and after a 3.5-hr recovery period interphase cells were analysed for MN. Using this time schedule only cells that were treated during mitosis or G2-phase plus mitosis were analysed. Four suspected aneuploidy-inducing agents (aneugens) were tested: griseofulvin, methyl 2-benzimidazole carbamate, diazepam and thiobendazole. All four compounds induced MN in the modified V79 assay. For further characterization of the modified V79/MN assay the clastogen mitomycin C (MMC) was investigated with respect to the time-dependency of MN induction. MMC did not induce MN in our modified V79 assay, but was positive after 14 hr of exposure. Induced MN were characterized by CREST staining and by measuring their size. The frequencies of CREST-positive and large MN were increased by the four suspected aneugens analysed. Furthermore, the use of the external metabolization systems, S-9 mix, and hepatocytes, was examined.

Genotoxicity of three-carbon compounds evaluated in the SCE test in vitro.

Three-carbon chemicals (chlorinated and nonchlorinated, saturated and unsaturated, hydroxy- and oxo-hydrocarbons) were assayed for genotoxicity. The sister chromatid exchange test in vitro served as the test system. Without S9 mix, the nonchlorinated solvents 1-propanol, 2-propanol, and 2-propanone (acetone) did not increase the SCE frequencies. All chlorinated 3-C hydrocarbons, except 1,2,3-trichloropropane, proved to be potent SCE inducers in V79 cells without S9 mix. In the presence of S9 mix, the results obtained with the nonchlorinated solvents were also negative, whereas 1,2,3-trichloropropane was transformed to SCE-inducing metabolites. The addition of S9 mix resulted in an increased SCE rate for 2,3-dichloropropanol, whereas genotoxicity of 2,3-dichloropropene, 1,2-dichloropropane, 1,3-dichloropropene, and 1,3-dichloropropanone was reduced. 1,3-dichloropropanol, 1,3-dichloropropene, and epichlorohydrin were substantially inactivated by S9 mix in the V79/SCE test. It can be concluded that the reactivity of the saturated dichloro compounds in the SCE test depends on the degree of oxidation. There is no general difference between the reactivity of alpha, beta-dichloro and alpha, omega-dichloro compounds.